Indicator for sepsis as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an indicator for sepsis as well as a preparation method and application of the indicator for sepsis, and comprises a kit for detecting sCD147 in body fluid, and the kit is a kit for quantitatively detecting sCD147 in body fluid based on a flow fluorescence technology. The kit comprises microspheres coupled with a capture antibody, a biotin-labeled detection antibody and a reporter molecule. Compared with the prior art, the indicator for sepsis as well as the preparation method and the application of the indicator can predict occurrence, development and prognosis of sepsis, and meanwhile, the invention provides the preparation method of the sCD147 detection kit which is simple to operate, high in sensitivity, good in repeatability and wider in application.

Description

[Technical field]

[0001] The present invention relates to the field of biomedical technology, and in particular to an indicator for sepsis and a preparation method and application thereof. [Background technology]

[0002] Sepsis is an infectious disease caused by bacterial or viral infection, which leads to an imbalance of the immune system and the release of a large number of inflammatory factors, eventually leading to multiple organ failure or even death. Most patients suffer from complications such as acute kidney injury, DIC, shock, etc., which are usually associated with higher morbidity and mortality. Therefore, accurate diagnosis and appropriate treatment of patients with sepsis are necessary conditions for achieving the best treatment effect.

[0003] Currently, the diagnosis and prognosis assessment of sepsis face two major challenges: first, the lack of simple 、 Rapid diagnostic markers, and secondly, the existing biomarkers for evaluating the prognosis of sepsis are insufficient. Although a series of plasma diagnostic markers for sepsis have been reported, including C-reactive protein (CRP) and procalcitonin (PCT), the diagnostic value of these markers is limited. At the same time, although the SOFA score is widely used, it is limited by its complexity and reliance on the subjective judgment of doctors. Given the complexity of sepsis diagnosis and prognosis assessment, it is crucial to develop new biomarkers to improve the accuracy of assessment. Therefore, it is necessary to provide a new biomarker and its preparation method to more accurately assess sepsis to solve the above-mentioned problems existing in the prior art. [Summary of the invention]

[0004] In order to overcome the above problems, the present invention proposes an indicator for sepsis and a preparation method and application thereof, which can effectively solve the above problems.

[0005] A technical solution provided by the present invention to solve the above technical problems is: to provide an indicator for sepsis and a preparation method and application thereof, including a kit for detecting sCD147 in body fluids, wherein the kit is a kit for quantitatively detecting sCD147 in body fluids based on flow fluorescence technology, and the kit includes microspheres coupled with capture antibodies, biotin-labeled detection antibodies and reporter molecules.

[0006] Preferably, the capture antibody is sCD147 rabbit monoclonal antibody, and the detection antibody is sCD147 mouse monoclonal antibody labeled with biotin.

[0007] Preferably, the detection antibody is selected to be an antibody that binds to a different antigen epitope corresponding to the capture antibody, so as to form a sandwich composite structure.

[0008] Preferably, the reporter molecule is streptavidin labeled with a fluorescein marker.

[0009] Preferably, the kit further comprises a calibrator, a quality control product 1, a quality control product 2, a sample diluent and a cleaning solution, wherein the calibrator, the quality control product 1 and the quality control product 2 are freeze-dried products containing sCD147 recombinant protein.

[0010] A method for preparing an indicator for sepsis comprises the steps of preparing materials, preparing microspheres coupled with capture antibodies, preparing biotin-labeled detection antibodies, preparing reporter molecules, preparing calibrators, preparing quality control products, preparing sample diluents, and preparing cleaning solutions.

[0011] Preferably, the preparation of microspheres coupled with capture antibodies comprises:

[0012] Take the microspheres, separate them magnetically, discard the supernatant, and add MES buffer to resuspend;

[0013] EDC and Sulfo-NHS were prepared into solutions using MES buffer;

[0014] Add EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence and mix well;

[0015] The microspheres were magnetically separated and the supernatant was discarded. The microspheres were washed once with MES buffer and magnetically separated again and the supernatant was discarded.

[0016] Add antibodies to the microspheres, supplement with MES buffer, and mix well;

[0017] Magnetic separation of microspheres, discarding the supernatant, adding blocking solution, mixing and blocking reaction;

[0018] The microspheres were separated by magnetic separation, the supernatant was discarded, and the preservation solution was added to resuspend and store.

[0019] Preferably, the preparation of the biotin-labeled detection antibody comprises dissolving activated biotin N-hydroxysuccinimide ester with anhydrous DMF, adding mouse anti-human sCD147 monoclonal antibody and incubating at room temperature, dialyzing with PBS solution overnight to remove unreacted biotin N-hydroxysuccinimide ester, and obtaining the biotin-labeled detection antibody.

[0020] Preferably, the reporter molecule is SA-PE, which is diluted to 5 μg / mL with PBS solution for later use.

[0021] An indicator for sepsis is used in the evaluation of sepsis.

[0022] Compared with the prior art, the indicator for sepsis of the present invention, its preparation method and application, compared with healthy people and non-sepsis patients, the plasma sCD147 level in sepsis patients is significantly increased, which can be used as a biomarker for the diagnosis of sepsis, and can predict the occurrence, development and prognosis of sepsis. At the same time, the present invention provides a preparation method of an sCD147 detection kit with simple operation, high sensitivity, good repeatability and wider application.

Brief Description of the Drawings

[0023] Figure 1 A distribution diagram of capture microspheres for indicators of sepsis and preparation methods and uses thereof of the present invention;

[0024] Figure 2 The sCD147 standard curve diagram of the indicator for sepsis and the preparation method and application thereof of the present invention;

[0025] Figure 3 This is a linear evaluation graph of sCD147 for the indicator for sepsis and the preparation method and application thereof of the present invention;

[0026] Figure 4 Schematic diagram of the level of plasma sCD147 in patients with sepsis;

[0027] Figure 5 This is a graph showing the correlation between plasma sCD147 levels and disease progression;

[0028] Figure 6 Schematic diagram showing that sCD147 significantly differentiates AKI, DIC and septic shock;

[0029] Figure 7 This is the prediction chart of sCD147 for patients' survival outcomes;

[0030] Figure 8 This is the prediction chart of sCD147 for the occurrence of sepsis;

[0031] Fig. 9 It is a correlation diagram between the test results of the kit of the present invention and the test results of ELISA. [Specific implementation method]

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and implementation examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] It should be noted that in the embodiments of the present invention, all directional indications (such as up, down, left, right, front, back, etc.) are limited to relative positions on the specified view, rather than absolute positions.

[0034] In addition, in the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0035] The level of soluble CD147 (sCD147) in plasma is closely related to the occurrence, development and prognosis of sepsis, and is expected to become a new marker for sepsis.

[0036] See also Figures 1 to 9 The indicator for sepsis of the present invention includes a kit for detecting sCD147 in body fluids, wherein the kit is a kit for quantitatively detecting sCD147 in body fluids based on flow fluorescence technology, and the kit includes microspheres coupled with capture antibodies, biotin-labeled detection antibodies and reporter molecules.

[0037] The microspheres have a particle size of 3-10 μm.

[0038] The capture antibody is sCD147 rabbit monoclonal antibody, which can recognize epitopes on human antigens that are not immunogenic in rodents, increasing the total number of targetable epitopes; rabbit monoclonal antibodies have a strong immune response to small molecules and haptens, which is not common in rodents; inbred rabbits are rarer, while most mouse strains are inbred, so rabbits have more immune response diversity; rabbits use a unique mechanism to genetically produce and diversify antibodies, giving them high affinity and specificity.

[0039] The detection antibody is a sCD147 mouse monoclonal antibody labeled with biotin. With the widespread use of more and more monoclonal antibody drugs in clinical practice, the occurrence of HAMA effect is gradually increasing. Interfering antibodies may cause false positive results by cross-linking capture antibodies and detection antibodies; they may also cause aggregation of detection antibodies by preventing the binding of antigens to detection antibodies, thereby causing false negative results. Based on this, rabbit monoclonal antibodies are selected as capture antibodies, and mouse monoclonal antibodies are selected as detection antibodies, which can effectively reduce the interference of HAMA effect.

[0040] The detection antibody selects an antibody that binds to a different antigen epitope corresponding to the capture antibody, and can form a sandwich composite structure of microsphere-capture antibody-antigen-detection antibody. The quantitative detection of the antigen is achieved by detecting the reporter molecule, which can greatly improve the accuracy and sensitivity of the detection.

[0041] The reporter molecule is streptavidin labeled with a fluorescein marker. Preferably, it is a fluorescent protein-based fluorescent substance, including: phycoerythrin (PE), allophycocyanin (APC), crosslinked allophycocyanin (crosslink-APC), Peridinium chlorophyll protein complex (PerCP), green fluorescent protein (GFP), etc. The binding mode of biotin and streptavidin has higher specificity and stability than other types of covalent coupling modes, making the binding of the reporter molecule and the detection antibody more stable and accurate, thereby effectively improving the accuracy of the detection results.

[0042] The kit also includes a calibrator, a quality control product 1, a quality control product 2, a sample diluent and a cleaning solution. The calibrator, the quality control product 1 and the quality control product 2 are freeze-dried products containing sCD147 recombinant protein.

[0043] The following Table 1 shows the composition of the kit:

[0044]

[0045]

[0046] The method for preparing an indicator for sepsis of the present invention comprises material preparation, preparation of microspheres coupled with capture antibodies, preparation of biotin-labeled detection antibodies, preparation of reporter molecules, preparation of calibrators, preparation of quality control products, preparation of sample diluents, and preparation of cleaning solutions.

[0047] The material preparation includes:

[0048] 6μm magnetic microspheres (concentration: 8×10 6 / mL, surface carboxyl activation);

[0049] capture antibody (rabbit anti-human sCD147 monoclonal antibody);

[0050] Detection antibody (mouse anti-human sCD147 monoclonal antibody);

[0051] Biotin N-hydroxysuccinimide ester;

[0052] SA-PE;

[0053] EDC;

[0054] Sulfo-NHS;

[0055] DMF;

[0056] PBS buffer (10 mM, pH 7.4);

[0057] Tris buffer (50 mM, pH 8.2);

[0058] MES buffer (50 mM, pH 6.0);

[0059] Blocking solution: Tris buffer + 1% BSA;

[0060] Preservation solution: PBS buffer + 0.1% BSA + 0.02% Tween-20 + 0.1% Proclin 300;

[0061] Magnets, etc.

[0062] The microspheres prepared by coupling with capture antibodies include:

[0063] Take 1.25 mL of microspheres, separate them magnetically, discard the supernatant, and add 0.16 mL of MES buffer to resuspend;

[0064] Weigh 5 and 50 mg of EDC and Sulfo-NHS respectively and prepare a 50 mg / mL solution with MES buffer;

[0065] Add 0.02 mL of EDC solution and 0.02 mL of Sulfo-NHS solution to the microsphere suspension in sequence and mix well; react at 25°C for 30 minutes;

[0066] The microspheres were magnetically separated and the supernatant was discarded. The microspheres were washed once with 0.2 mL of MES buffer and magnetically separated again and the supernatant was discarded.

[0067] Add 50 μg of antibody to the microspheres, add MES buffer to 0.2 mL, and mix well; react at 25°C for 3 hours;

[0068] Magnetic separation of microspheres, discard the supernatant, add 0.4 mL of blocking solution, mix and block the reaction at 25°C for 12-18 hours;

[0069] Microsphere magnetic separation, discard the supernatant, add 100mL preservation solution to resuspend, and store at 4℃.

[0070] Test on a flow cytometer or flow fluorimeter, refer to Figure 1 .

[0071] The preparation of the biotin-labeled detection antibody includes dissolving 10 mg of activated biotin N-hydroxysuccinimide ester with 1 mL of anhydrous DMF, taking 10 μL, adding 200 μg of mouse anti-human sCD147 monoclonal antibody (detection antibody), incubating at room temperature for 1 hour, and dialyzing with 100 mL of PBS solution overnight to remove unreacted biotin N-hydroxysuccinimide ester, thereby obtaining the biotin-labeled detection antibody.

[0072] The reporter molecule preparation includes the reporter molecule being SA-PE, which is a commercially available product and is diluted to 5 μg / mL with a PBS solution for later use.

[0073] The preparation of the calibration product includes adding fetal bovine plasma as a matrix to the calibration product to eliminate the matrix effect, the matrix is ​​fetal bovine plasma (20% v / v), BSA (0.1% w / v) PBS (10mmol / L), proclin300 (0.2% v / v), adding recombinant sCD147 antigen, mixing, lyophilizing and setting aside for use. It is used for establishing the calibration curve of the kit.

[0074] The quality control product is prepared, including two levels of high and low values, and fetal bovine plasma is added as a matrix to eliminate the matrix effect. The matrix is ​​fetal bovine plasma (20% v / v), BSA (0.1% w / v) PBS (10 mmol / L), and proclin300 (0.2% v / v). Low and high values ​​of recombinant sCD147 antigens are added respectively, mixed, and freeze-dried for use.

[0075] The sample diluent is prepared, and the sample diluent contains human negative plasma (20% v / v), BSA (0.1% w / v), PBS (10 mmol / L), and proclin300 (0.2% v / v).

[0076] The preparation of the cleaning solution comprises the cleaning solution containing BSA (0.1% w / v), PBS (10 mmol / L), proclin300 (0.1% v / v), and Tween 20 (0.2‰ v / v).

[0077] The method of using the kit of the present invention is as follows:

[0078] Step Y1, preparation of calibrators: add 1 mL of purified water to the calibrators in the kit, mix well, and perform gradient dilution with sample diluent to obtain calibrators of different concentrations for the establishment of a standard curve.

[0079] Step Y2, preparation of quality control products, take two quality control products in the kit, add 1 mL of purified water respectively, mix well, and obtain two quality control products of different concentrations for quality control evaluation of the kit.

[0080] Step Y3, take 50 μL of calibrator / quality control / plasma sample to be tested, add 50 μL of capture antibody microspheres, mix well, and incubate at 37°C for 30 minutes.

[0081] Step Y4, after magnetic separation of the capture microspheres, wash with washing solution three times, add 50 μL of biotin-labeled detection antibody, add 50 μL of SA-PE, mix well, and incubate at 37°C in the dark for 30 min.

[0082] Step Y5, after magnetic separation, the captured microspheres are washed three times with a washing solution and detected using a flow cytometer or a flow fluorimeter.

[0083] In step Y6, the signal of phycoerythrin (PE) is read in the fluorescence channel by a flow cytometer or a flow fluorometer, and the signal reported by phycoerythrin (PE) represents the total sCD147 concentration.

[0084] The test results and analysis of the kit of the present invention include:

[0085] 1. Establishment of standard curve

[0086] Use the calibrator provided in this kit to dilute the calibrator to different concentrations to obtain calibrators of different concentrations. Figure 2 The standard curve of

[0087] 2. Linear Evaluation

[0088] Select a high-concentration sample at the upper limit of the linear range and dilute it into at least 7 samples of different concentrations (xi). Test each concentration 3 times and calculate the mean value (yi) of the test results. Use the dilution concentration (xi) as the independent variable and the mean value (yi) of the test results as the dependent variable to calculate the linear regression equation. Calculate the correlation coefficient (R) of the linear regression. The results are shown in Figure 3 According to the formula, the correlation coefficient R is greater than 0.99, which shows a good correlation.

[0089] 3. Blank Limit and Detection Limit Assessment

[0090] Blank limit: Use sample diluent, repeat the test 20 times, and calculate the average and standard deviation (SD), and calculate x ± 2SD signal value. Substitute this value into the standard curve equation of the kit to obtain the corresponding sample concentration value, which is the blank limit of this kit.

[0091] Detection limit: Test 5 samples with low concentrations close to the detection limit (the approximate detection limit is estimated based on the blank limit value, which is slightly higher than the blank limit). Test each sample 5 times and sort the test results by size. The number of test results below the blank limit value should be less than or equal to 3.

[0092] As shown in Table 2, the blank limit is 1.21 pg / mL and the detection limit is 2.45 pg / mL, which has high sensitivity.

[0093] Table 2. Blank limit and detection limit (pg / mL)

[0094]

[0095] 4. Repeatability Evaluation

[0096] Select high and low value quality control products and perform repeatability tests.

[0097] In one day, the test was performed 10 times in parallel and the CV was calculated;

[0098] As can be seen from Table 3, the repeatability CV of low and high value quality control is less than 5%

[0099] Table 3. Repeatability (pg / mL)

[0100]

[0101] 5. Clinical Evaluation

[0102] 1. Materials and methods

[0103] 1. Patients diagnosed with sepsis according to the Sepsis 3.0 diagnostic criteria are excluded from the plasma collection of the present invention if they have blood diseases, novel coronavirus infection, long-term antibiotic treatment, or combined autoimmune diseases.

[0104] The present invention tested the frozen plasma of 48 healthy people and 220 ICU patients who met the criteria, of which 72 had acute kidney injury, 37 had DIC, 103 had septic shock, and 54 died. At the same time, the frozen plasma of 60 patients admitted to the hospital for infection in the emergency department was tested, of which 24 developed sepsis later.

[0105] 2. SOFA score for patients with sepsis: calculated by clinicians according to the regulations of the European Society of Intensive Care Medicine.

[0106] 3. Electronic data processing was used to record clinical and laboratory indicators and organ or system involvement data, including demographic information, age, gender, and laboratory indicators of cytokines such as IL-6. At the same time, whether acute kidney injury (AKI), DIC, and shock occurred during hospitalization was also recorded.

[0107] 4. Measurement of sCD147: The level of plasma sCD147 in sepsis patients was measured according to the operating procedures of the ELISA manual.

[0108] Statistical analysis

[0109] SPSS software and GraphpadPrism9.5 were used for statistical analysis. The two independent samples were compared by Mann-Whitney test, and Spearman rank correlation test analysis was used to calculate the correlation between two variables. Receiver operating characteristic curve (ROC curve) analysis was applied to evaluate the sensitivity and specificity of sCD147 indicative function. The optimal cutoff point of sCD147 was selected according to the maximum value of Youden index. Calibration curves and decision curves were used to evaluate the predictive value. All statistical analyses with p value <0.05 were considered statistically significant.

[0110] (II) Results and analysis

[0111] 1. Elevated plasma sCD147 levels in patients with sepsis

[0112] Compared with healthy controls and non-septic patients, the level of sCD147 was significantly increased in septic patients ( Figure 4 A). To clarify whether the level of sCD147 can be used as an indicator of sepsis patients, ROC analysis was performed to distinguish sepsis patients from healthy and non-septic patients. Compared with healthy subjects, the AUC reached 1, and compared with non-septic patients, the AUC was 0.83 ( Figure 4 BC), the calibration curve illustrates the accuracy of the discrimination ability ( Figure 4 D), the clinical decision curve shows that using the level of sCD147 to distinguish between sepsis and non-sepsis patients can benefit patients ( Figure 4 E).

[0113] 2. Correlation between sCD147 levels and clinical manifestations

[0114] The correlation between plasma sCD147 levels and clinical characteristics was also evaluated by Spearman correlation. sCD147 levels were significantly positively correlated with SOFA scores of disease severity, IL-6, IL-10, and PCT, as well as with renal function indicators creatinine and urea levels, and coagulation indicators PT and APTT levels ( Figure 5 ).

[0115] 3. Increased plasma sCD147 levels can distinguish whether patients have acute kidney injury (AKI), DIC and shock.

[0116] The present invention compared the levels of plasma sCD147 in sepsis patients with different clinical manifestations and found that the level of sCD147 was significantly increased in patients with acute kidney injury, DIC and shock ( Figure 6 AC), and the ROC curves were used to distinguish non-acute kidney injury from kidney injury, non-DIC from DIC, and no shock from shock. The AUC of sCD147 for distinguishing AKI was 0.736, and the AUC of sCD147 and creatinine for distinguishing AKI was 0.8 ( Figure 6 D), the AUC of sCD147 for distinguishing DIC was 0.736, and the AUC of sCD147 combined with commonly used clinical coagulation indicators for distinguishing DIC was 0.79 ( Figure 6 E). In addition, the AUC of sCD147 for distinguishing shock was 0.733. Based on binary logistics regression, the AUC of sCD147 combined with IL-6 and SOFA score for distinguishing shock was 0.852 ( Figure 6 F)

[0117] 4. Plasma sCD147 levels can predict patient survival and death

[0118] Compared with survivors, sCD147 levels were significantly increased in non-survivors ( Figure 7 A), the generated ROC curve was used to evaluate the ability of sCD147 to predict survival and death. The AUC of sCD147 for predicting death was 0.755, which was better than SOFA score ( Figure 7 B). The calibration curve is used to evaluate the accuracy of its prediction ( Figure 7 C), DCA analysis showed that it can benefit patients in clinical application ( Figure 7 D) Patients with high levels of sCD147 were more likely to die at 30 and 90 days ( Figure 7 EF).

[0119] 5. Plasma sCD147 levels can predict the occurrence of sepsis

[0120] The present invention tested the plasma of 60 patients admitted to the emergency department. Compared with patients with only infection, the level of sCD147 was significantly increased in patients who subsequently developed sepsis ( Figure 8 A), the AUC of plasma sCD147 level in predicting the occurrence of sepsis was 0.88 ( Figure 8 B), the calibration curve is used to evaluate the accuracy of the prediction ( Figure 8 C), DCA analysis showed that it has good value in clinical application ( Figure 8 D).

[0121] 6. The results of ELISA method are positively correlated with the results of our invented kit

[0122] The method for detecting sCD147 in plasma proposed by the present invention has a positive correlation between the detection result and the sCD147 concentration value detected by the widely accepted and used ELISA method ( Fig. 9 ).

[0123] The use of the indicator for sepsis of the present invention in sepsis assessment comprises the following steps:

[0124] Step S1, adding microspheres coated with sCD147 capture antibody to the plasma sample, mixing, and incubating;

[0125] Step S2, adding biotin-labeled sCD147 detection antibody and fluorescein-labeled streptavidin to the plasma sample in step S1, mixing, and incubating in the dark;

[0126] Step S3, detecting the plasma sample in step S2 using a flow cytometer or a flow fluorimeter;

[0127] Step S4, the signal of the fluorescent protein is read on a flow cytometer or a flow fluorometer, and the signal reported by the fluorescent protein represents the total sCD147 concentration.

[0128] Compared with the prior art, the indicator for sepsis of the present invention, its preparation method and application, found that the plasma sCD147 level was significantly increased in sepsis patients and was related to the clinical symptoms of the disease. Therefore, the detection of sCD147 in plasma can provide an important reference for auxiliary diagnosis, medication guidance, efficacy monitoring and prognosis evaluation of sepsis; based on this, the present invention provides a kit for detecting sCD147 in body fluids, which is used to guide auxiliary diagnosis, medication guidance, efficacy monitoring and prognosis evaluation of sepsis; compared with the prior art, the selected flow fluorescence technology and rabbit monoclonal antibody of the kit provided by the present invention are helpful to eliminate cross-influence and improve the sensitivity and accuracy of the detection results; the kit provided by the present invention has a detection sensitivity of sCD147 of 2.4pg / mL and a repeatability CV of <6%, which is more conducive to wide clinical application and has the advantages of simple operation, high sensitivity, good repeatability and wider clinical application.

[0129] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any modifications, equivalent substitutions and improvements made within the concept of the present invention should be included in the patent protection scope of the present invention.

Claims

1. An indicator for sepsis, characterized in that The invention comprises a kit for detecting sCD147 in body fluids, wherein the kit is a kit for quantitatively detecting sCD147 in body fluids based on flow fluorescence technology, and the kit comprises microspheres coupled with capture antibodies, biotin-labeled detection antibodies and reporter molecules.

2. The indicator for sepsis according to claim 1, characterized in that The capture antibody is sCD147 rabbit monoclonal antibody, and the detection antibody is sCD147 mouse monoclonal antibody labeled with biotin.

3. The indicator for sepsis according to claim 1, characterized in that The detection antibody selects an antibody that binds to a different antigen epitope corresponding to the capture antibody, and a sandwich composite structure can be formed.

4. The indicator for sepsis according to claim 1, characterized in that The reporter molecule is streptavidin labeled with fluorescein.

5. The indicator for sepsis according to claim 1, characterized in that The kit also includes a calibrator, a quality control product 1, a quality control product 2, a sample diluent and a cleaning solution. The calibrator, the quality control product 1 and the quality control product 2 are freeze-dried products containing sCD147 recombinant protein.

6. A method for preparing an indicator for sepsis, characterized in that: It includes material preparation, preparation of microspheres coupled with capture antibodies, preparation of biotin-labeled detection antibodies, preparation of reporter molecules, preparation of calibrators, preparation of quality control products, preparation of sample diluents, and preparation of cleaning solutions.

7. The method for preparing an indicator for sepsis according to claim 6, characterized in that: The microspheres prepared by coupling with capture antibodies include: Take the microspheres, separate them magnetically, discard the supernatant, and add MES buffer to resuspend; EDC and Sulfo-NHS were prepared into solutions using MES buffer; Add EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence and mix well; The microspheres were magnetically separated and the supernatant was discarded, and the microspheres were washed once with MES buffer and magnetically separated again and the supernatant was discarded; Add antibodies to the microspheres, supplement with MES buffer, and mix well; Magnetic separation of microspheres, discarding the supernatant, adding blocking solution, mixing and blocking reaction; The microspheres were separated by magnetic separation, the supernatant was discarded, and the preservation solution was added to resuspend and store.

8. The method for preparing an indicator for sepsis according to claim 6, characterized in that: The preparation of the biotin-labeled detection antibody comprises dissolving activated biotin N-hydroxysuccinimide ester with anhydrous DMF, adding mouse anti-human sCD147 monoclonal antibody and incubating at room temperature, dialyzing with PBS solution overnight, removing unreacted biotin N-hydroxysuccinimide ester, and obtaining the biotin-labeled detection antibody.

9. The method for preparing an indicator for sepsis according to claim 6, characterized in that: The reporter molecule is SA-PE, which is diluted to 5 μg / mL with PBS solution for later use.

10. Use of the indicator for sepsis according to any one of claims 1 to 5 in sepsis assessment.

Images